Special problems are presented in the biochemical reactions used to assay a specific composition in a mixture of materials, separate one material from another, or catalyze a reaction using an enzyme. Frequently, the chemicals or molecules useful for such assays, separations, or reactions are scarce and costly. If such biologically active materials are not attached to a support material by some means, the scarce and costly materials will be lost for further usage, repeat experiments, and the like.
Therefore, in the biochemistry arts, methods involving a sequence of processing steps have been developed to immobilize various biologically active materials to supports where the supports have been formed in the absence of biologically active materials. Generally, such methods of immobilization may be divided into the following categories: covalent bonding or crosslinking of the biologically active material to a support; adsorption onto a support; or inclusion in a polymer matrix wherein the biologically active material is trapped or encapsulated in the lattice of the polymer. (A. Hoffman, Radiat. Phys Chem. 18(1,2), 323-342 (1981)).
However, depending upon the application, immobilization by covalent bonding or crosslinking may damage the biologically active material to the extent that its activity is reduced or lost. (K. Buchholz et al., Meth. Enzymol. 135, 3-30 (1987)). Even if the biologically active material retains its biological activity, experience has shown in production of covalently bonded immobilized materials that the distribution of biologically active materials may not be uniform.
Adsorption of such materials to a surface often results in the loss of activity. Further, adsorption is often a reversible process such that the biologically active material can be lost from the support by equilibration with the reaction media which may cause undesired contamination of the reaction products. (J. Andrade in "Surface and Interfacial Aspects of Biomedical Polymers, Volume II Protein Adsorption", Chapter 1, J. Andrade Ed., Plenum Press, New York (1985)).
Inclusion of the biologically active material by entrapment or encapsulation has previously been a preferred method for enzymes since the method relies upon the physical trapping of a large molecule in a polymer matrix from which it is very slow to escape. (A. Hoffman, referenced above). For materials which interact with the entrapped molecule and which are small enough to diffuse through this matrix, these methods are satisfactory, but for large reaction materials such as large proteins, the matrix prevents enzyme reactions.
Further, entrapment methods conventionally employ gel matrices formed by the polymerization of hydrophilic monomers and crosslinkers to form hydrogels. (U.S. Pat. No. 3,788,950 and K. O'Driscoll et al., Biotech. Bioeng. 14, 849-850 (1972)). These hydrogels are typically mechanically weak and may not be able to withstand the rigors of repeated usage without breaking up. Thus, the biologically active material and its matrix may be lost during the assay, separation, or reaction.
Encapsulations have been performed using gamma irradiation to polymerize hydrophilic monomers in order to trap enzymes, but such radiation polymerization has required the freezing of the enzyme/monomer mixture to encapsulate the enzymes therein. (I. Kaetsu et al., Radiat. Phys. Chem. 14, 595-602 (1979)). Thus, the immobilization of an enzyme in a hydrophilic matrix by this process requires excessive energy. Further, using gamma radiation of high dosages could destroy the biological activity of the enzyme. (E. Galas et al., Radiochem. Radioanal. Let. 43(6), 355-362 (1980)).
Hydrophobic monomers have also been used to entrap enzymes by polymerization of frozen mixtures or dispersions. These processes result in the preparation of beads as the hydrophobic monomers separate into droplets as the water freezes. (I. Kaetsu et al., Biotech. Bioeng. 21, 863-873 (1979)).
Unfortunately, the prior methods of encapsulation or entrapment of biologically active materials do not allow the immobilized species to bind or react with large molecules while retaining the species within the matrix. Encapsulation or entrapment would not allow for the immobilized species to interact with both large and small molecules.
In other words, most of the methods for direct incorporation of biologically active materials into the support relate to supports such as beads, gels, and particles.
Immobilization of biologically active materials onto the surfaces of membrane supports is known, but the active materials are introduced after membrane formation. (European Patent Office, Publication 0 294 186). This can lead to problems of leakage of the biologically active material, or non-uniform distribution of the biologically active material on the support, or require additional processing steps. Membrane supports are particularly desirable for bioseparations, enzyme-catalyzed reactions and the like.
Conventional processes have also been developed for the preparation of emulsions in the presence of biologically active materials. Such emulsions may be either water in oil (W/O) or oil in water (O/W) or other combinations of hydrophilic/hydrophobic liquids. (U.S. Pat. No. 4,774,178).
With the use of some surfactants, biologically active materials may be dispersed into an emulsion which qualifies as a microemulsion because of the microscopic extent of dispersion of the oil and water constituents. (P. L. Luisi, Angew. Chem. Int. Ed. Engl. 24(6), 439-528 (1985)). When the oil and water components of the microemulsion are so dispersed that it is not possible to characterize the microemulsion as either a water in oil microemulsion or an oil in water microemulsion, a bicontinuous microemulsion is recognized. (L. E. Scriven, Nature 263, 123 (1976)).
Thus, the water and oil components of the microemulsion are so intermixed that both components are intimately mixed in continuous contact with like material. A suitable analogy for a bicontinuous microemulsion is a water saturated sponge. Both the water and the sponge components are intimately mixed but each is also in continuous contact with its like material.
Previously processes have used microemulsions to prepare polymers and beads. (J. O. Stoffer et al., J. Dispersion Sci. and Technol. 1(4), 393-412 (1980) and U.S. Pat. No. 4,521,317). However, the polymerization of microemulsions containing biologically active materials has only been used to prepare nanometer-sized particles. (U.S. Pat. No. 4,021,364). Films and membranes, most desired for biological reactions such as assays, separations and enzyme-catalysis, have not been produced by microemulsion techniques.
Thus, what is needed is a polymer formed having biologically active or reactive sites at its surfaces in order to enable biochemical usage. What is also needed is a method of preparing the polymer using a low cost emulsion method which does not harm the bioreactivity of the biologically active material.